Fe(II)Cl2 amendment suppresses pond methane emissions by stimulating iron-dependent anaerobic oxidation of methane

Abstract Aquatic ecosystems are large contributors to global methane (CH4) emissions. Eutrophication significantly enhances CH4-production as it stimulates methanogenesis. Mitigation measures aimed at reducing eutrophication, such as the addition of metal salts to immobilize phosphate (PO43−), are now common practice. However, the effects of such remedies on methanogenic and methanotrophic communities—and therefore on CH4-cycling—remain largely unexplored. Here, we demonstrate that Fe(II)Cl2 addition, used as PO43- binder, differentially affected microbial CH4 cycling-processes in field experiments and batch incubations. In the field experiments, carried out in enclosures in a eutrophic pond, Fe(II)Cl2 application lowered in-situ CH4 emissions by lowering net CH4-production, while sediment aerobic CH4-oxidation rates—as found in batch incubations of sediment from the enclosures—did not differ from control. In Fe(II)Cl2-treated sediments, a decrease in net CH4-production rates could be attributed to the stimulation of iron-dependent anaerobic CH4-oxidation (Fe-AOM). In batch incubations, anaerobic CH4-oxidation and Fe(II)-production started immediately after CH4 addition, indicating Fe-AOM, likely enabled by favorable indigenous iron cycling conditions and the present methanotroph community in the pond sediment. 16S rRNA sequencing data confirmed the presence of anaerobic CH4-oxidizing archaea and both iron-reducing and iron-oxidizing bacteria in the tested sediments. Thus, besides combatting eutrophication, Fe(II)Cl2 application can mitigate CH4 emissions by reducing microbial net CH4-production and stimulating Fe-AOM.


Introduction
Aquatic ecosystems are responsible for half of the global CH 4 emissions (Rosentreter et al. 2021 ).CH 4 release from organic sediments is str ongl y driv en by eutr ophication (Davidson et al. 2018 ), whic h r emains a significant and ongoing worldwide pr oblem (Beaulieu et al. 2019 ).Eutrophication increases autochthonous or ganic matter pr oduction, whic h pr ecludes substr ate limitation and promotes methanogenesis, and hence is estimated to increase CH 4 emissions by 30%-90%, making eutrophic waters hotspots for CH 4 emission (Attermeyer et al. 2016, Beaulieu et al. 2019 ).Besides nutrient-load reduction, geo-engineering techniques are increasingly used to combat eutrophication.These include techniques using PO 4 3 − (P)-binding compounds such as F e(II)Cl 2 , lanthanum-modified bentonite cla y, and aluminummodified zeolite (Jan čula and Maršálek 2011 ).Howe v er, ther e is little insight into how eutrophication remediation strategies affect aerobic and anaerobic CH 4 -cycling microorganisms, and how these affect CH 4 emissions (Jan čula and Maršálek 2011 , Nijman et al. 2022 ).
Ther efor e, in this study, we tested the effect of the P-binding agent Fe(II)Cl 2 on microbial CH 4 -cycling in field experiments and batch incubations.In oxic layers of Fe-rich sediments the majority of the phosphorus (P) is bound to ferric iron (Fe(III)) (Parsons et al. 2017 ).Once the bound Fe(III)-P r eac hes the anoxic zone of the sediment, bound P is released from the Fe(III)-P complex, as a consequence of the reduction and accompanying dissolution of the Fe particles (Cooke et al. 1993 ).Subsequently, in the anoxic zone of aquatic sediments, in the presence of Fe(II), P is known to form vivianite (Fe(II) 3 (PO 4 ) 2 r 8 H 2 O) through authigenesis.Vivianite is a hydr ated ferr ous phosphate miner al that immobilizes phosphate (Walpersdorf et al. 2013, Rothe et al. 2014, Liu et al. 2018, Heinrich et al. 2021 ).Ho w e v er, when exposed to alternativ e electr on acceptors (e.g.O 2 , NO 3 − , NO 2 , SO 4 2 − ) up to 50% of the Fe(II) present in vivianite can be oxidized to poorly crystalline mixed-valence or ferric F e(III)-P molecules , increasing the bioa vailable F e(III) in the sediment (Nielsen and Nielsen 1998, Miot et al. 2009, Kusunoki et al. 2015, Rothe et al. 2016 ).Additionall y, ferr ous Fe-salts like Fe(II)Cl 2 that ar e dir ectl y a pplied in the sediments can be dir ectl y oxidized with O 2 or NO 3 − (Benz et al. 1998, Nielsen and Nielsen 1998, Oikonomidis et al. 2010 ).Fe(II)Cl 2 application could therefor e, thr ough dir ect Fe(II)Cl 2 oxidation or through vivianite oxidation, substantiall y incr ease the bioav ailable Fe(III) concentr ations in the sediment.CH 4 formation (methanogenesis) is a microbial process commonly taking place in anoxic sediments (Segers 1998 ).CH 4 can be oxidized to CO 2 by methanotrophic microorganisms, either aerobicall y, using O 2 as electr on acceptor, or anaer obicall y, using differ ent alternativ e electr on acceptors (e .g. NO 3 − , F e(III), Mn Fe-AOM is mediated by anaerobic CH 4 -oxidizing archaea (ANME), which belong to three distinct clades (ANME 1-3) (Weber et al. 2017 ).Members of the ANME-1 clade are highly diverse, and fall in the order of Methanophagales .ANME-2 is comprised of three families, all in the order of Methanosarcinales , of which one is observed in freshwaters; the Methanoperedenaceae (ANME-2d), and two ar e observ ed in marine systems: Methanocomedenaceae and Methanogasteraceae .ANME-3 is closel y r elated to known methanogens, and r epr esents a nov el genus within the famil y of Methanosar cinaceae (Chad wick et al. 2022 ) .
Since Fe(II)Cl 2 addition potentially leads to elevated Fe(III) concentrations in the sediment, it is hypothesized that this could lead to a significant change in the contribution of Fe-AOM to the CH 4 cycle in the long term, lowering both internal PO 4 3 − concentrations and in situ CH 4 emissions.
Here we aim to unravel the effects of Fe(II)Cl 2 addition on the aquatic carbon cycle.With this study we generate new insights into how bioremediation techniques -besides combating eutrophication-can help in reducing aquatic CH 4 emissions, contributing to climate-smart water management.

Field description
To e v aluate the effect of Fe(II)Cl 2 on CH 4 dynamics, an in-situ enclosure experiment was established inside an eutrophic pond.The pond is located at the property of Water Authority 'Brabantse Delta', Breda, The Netherlands (51 • 33 45.5 N 4 • 46 58.7 E).The pond consists of an open water system, which is connected to a surr ounding c hannel system and encompasses a water surface area of roughly 14 500 m 2 , with an average water depth of 1.1 m (Kang et al. 2023 ).On av er a ge, the total water N and P concentrations range from 0.46 to 4 mg N L −1 and 0.03-0.22mg P L −1 and the bioav ailable P fr action (top 6 cm sediment) was on av er a ge 0.91 mg g dw -1 .More details on site description can be found in Kang et al. ( 2023 ).

Experimental setup
In March 2020, a total of 8 tr anspar ent P erspex c ylindrical enclosures (1.05 m diameter, 1.30 m height and a total volume of 865 L, open at the top and bottom) were firmly pushed into the ponds' sediment (a ppr oximatel y 0.2 m deep) (Kang et al. 2023 ).The enclosur es wer e positioned in line, at a 30 cm interv al, and the topedge of the enclosures extended approximately 25 cm above the water surface.After placement (9 th March 2020), the enclosures were left to settle for approximately 1.5 months.On the 8 th of April 2020 the enclosures were randomly treated with Fe(II)Cl 2 (N = 4) or left as a control (N = 4) (Kang et al. 2023 ).While the contr ol enclosur es wer e left undisturbed, the Fe(II)Cl 2 tr eatment r eceived 19.1 g of crystalline F e(II)Cl 2 (C AS-Nr.: 13478-10-9, Honeywell) that was dissolved in 200 ml anoxic acetate buffer, to obtain a 0.48 M Fe(II)Cl 2 -solution (pH = 4.2).In total 20 ml of this solution was dir ectl y injected into the upper 6 cm of the sediment at 10 differ ent spots, whic h wer e r andoml y c hosen fr om a 16-squar ed grid placed on top of each enclosure (Kang et al. 2023 ).The applied dosage of Fe(II)Cl 2 was based on the amount of bioavailable P in the first 6 cm of the sediment, to target a 1.5 molar ratio (Fe: P), and is further described in Kang et al. ( 2023 ).In the Fe(II)-treated sediments , F e(III) accounted for between 66% and 91% of the total F e , in the control it was 81% (Kang et al. 2023 ).

Field measurements
To quantify the effect of the Fe(II)Cl 2 treatment on the CH 4 emissions from the pond enclosures, we measured CH 4 emissions (totaling 5 sampling occasions) from the 15 th of September 2021 (17 months after Fe(II)Cl 2 addition) until the 10 th of November 2021.We measured CH 4 emission after over a year of Fe(II)Cl 2 application because in general differences in long-term ecosystem effects become a ppar ent after a complete gr owing season, due to indirect effects of P-binding agents on the pond-community, for example by changing macrophyte development (Nijman et al. 2022 ).During the measuring period, O 2 concentrations 1 cm above the sediment-water interface ranged between 0.2-6.3mg L −1 for contr ol enclosur es , and between 0.1 and 8.2 mg L −1 in F e(II)Cl 2 -treated enclosur es.Additionall y, the diffusiv e CH 4 emissions fr om the enclosur es wer e measur ed biweekl y.To limit potential CH 4 -emission variation caused by measurement time, all measurements were performed around midday, between 11:00 and 14:00 h, hence we did not account for possible diel v ariability.Fluxes wer e measur ed using the floating chamber method, and ebullition was measured by permanently installed bubble tr a ps (Almeida et al. 2016 ).The bubble tr a ps consisted of up-side down funnels (surface ar ea of 0.033 m 2 ) connected to glass (1000 ml) collection bottles filled with pond water as exemplified in Almeida et al. ( 2016 ).During the study duration no bubbles were caught, indicating ebullition was of minor importance .T he diffusive flux was measured using a tr anspar ent closed floating chamber (height 30 cm, diameter 28.8 cm) that was placed on top of the water column of the enclosure .T he chamber was connected to an Ultra-portable greenhouse gas analyzer (UGGA-Los Gatos ®) that was used to quantify the change in CH 4 concentration over a period of 3 min, using a closed loop system (Almeida et al. 2016 ).After placing the chamber, w e w aited for a ppr oximatel y 1 min for the flux to become stable, to ensure we were not underestimating or overestimating the CH 4 flux.The UGGA has an oper ating r ange of 0.01-100 ppm for CH 4 , with a 10-second response time, and a measuring frequency of 1 Hz.Although wind speed is a known driver of water-air fluxes of CH 4 , due to its effect on turbulent mixing, wind effects were limited because of the enclosur e-edges pr e v ented wind-driv en wa ve action.T he flux was calculated using equation 2 .Where F is the gas flux (mg m −2 d −1 ), V is chamber volume (L), A is the area of the chamber surface (m 2 ), ' slope ' is the CH 4 concentr ation c hange ov er time (ppm s −1 ); P is the atmospheric pr essur e (atm), T is the temper atur e in ( o K); R is the gas constant = 8.205746 × 10 −5 m 3 atm mol −1 K −1 ; F 1 is the molecular weight of CH 4 (16 g mol −1 ); F 2 is the conversion from seconds to da ys .At the end of the experiment, 3 sediment cores (diameter 6 cm, height 60 cm) were collected in eac h enclosur e using a UWITEC sediment corer (UWITEC, Mondsee , Austria).T he sediment of one core was sliced every 1 cm and analyzed for total P and total F e .The other cores were used for batch incubations.

Incubation experiments
Four sets of incubation experiments were performed (Fig. 1 ).To e v aluate the effect of the Fe(II)Cl 2 treatment in the enclosures, enclosur e sediments wer e incubated under oxic and anoxic conditions allowing for rate measurements of net CH 4 -production and CH 4 -oxidation.First, the aerobic top layer of the sediment cores (first 2 cm), as observed by a change in sediment color from light (oxidized) to black (anoxic), was used to quantify the aerobic CH 4oxidation rate, and subsequently to determine sediment moisture content by drying the sediments for 3 days at 70 • C. Next, another intact sediment core was tr ansferr ed to an anaerobic chamber ( < 10 ppm O 2 ), where the top 2 cm was remo ved.T he la y er betw een 2 and 6 cm sediment-depth was used to quantify sediment CH 4 -production and potential Fe-dependent anaerobic CH 4 -oxidation (incubations 2, 3 and 4).
To measure potential aerobic CH 4 -oxidation rates, 5 g of sediment + 10 ml of oxygenated ( ∼7 mg L −1 ) enclosure water was incubated under atmospheric (oxic) conditions in 120 ml gastight serum bottles.After sediment transfer, the bottles were capped and injected with 1.2 ml ( = 1%) 99% CH 4 gas, as in Nijman et al. ( 2022 ).The sediments were incubated at 18 • C and mixed continuously using a gyratory shaker (105 r/m).The CH 4 concentration in the headspace was measur ed dir ectl y after injection of 1.2 ml ( = 1%) CH 4 , and daily at 10:00, 13:00, 17:00 h during 4 da ys , using a gas c hr omatogr a ph equipped with flame ionization detection (He wlett P ac kard HP 5890 Series II Gas Chr omatogr a ph, Agilent Tec hnologies, California, USA).To ensur e the incubation bottles did not become anoxic, we measured O 2 concentrations in all the bottles using a Microx TX3 oxygen microsensor (flat-broken needle tip, Pr esens, German y), at the end of the incubation experiments.
To determine net CH 4 -production rates ( = CH 4 -productionanaer obic CH 4 -oxidation), dir ect anaer obic CH 4 -oxidation r ates and potential Fe-AOM (all corrected for 82% sediment moisture content), we incubated 25 g of sediment (2-6 cm depth) + 15 ml anoxic enclosure water in 140 ml gastight serum bottles under fully ano xic conditions.Ano xia was confirmed using Microx TX3 oxygen microsensors (flat-broken needle tip, Presens, Germany) in all bottles.For these anoxic incubations, different treatments were used: 1) 'H 2 O'-containing only sediment and water, used to determine the net CH 4 -production under control conditions; 2) 'CH 4 '-supplemented with 10 ml of 99% 13 CH 4 ; and 3) 'CH 4 + Fe'-supplemented with 10 ml of 99% 13 CH 4 and 3 mM ferrihydrite (Fe(III) 2 O 3 •0.5H 2 O) to determine potential rates of Fe-AOM.The bottles were k e pt in a climate-controlled room at 18 • C in the dark.Throughout 129 days, a ppr oximatel y once per month the total CH 4 , 13 CO 2 and 12 CO 2 concentrations of the headspace were determined r epeatedl y by gas c hr omatogr a phy coupled to mass spectr ometry (Tr ace DSQ II, Thermo Finnigan, Austin TX, USA).The concentration of CH 4 was quantified as described for aerobic CH 4 -oxidation.Due to simultaneous CH 4 -production and CH 4consumption in anoxic incubations, the AOM potential was monitored by the increase in 13/12 CO 2 rather than the change in CH 4 concentration.To test for the potential of Fe-AOM in these incubations, we sim ultaneousl y measur ed the dissolv ed F e(II), total F e(II) and total Fe in water and sediment using a colorimetric ferrozine assay (Schaedler et al. 2018 ).

16S rRNA sequencing
To explore the microbial potential for Fe-AOM we extracted DNA from the original in situ enclosure sediment which was used for the anaerobic incubations (2-6 cm) and from the anoxic batch incubations at the end of the 130-day incubation period.DNA was extracted using the Po w erSoil DN A extraction kit (DNeasy Po w erSoil Pr o Kit, QIAGEN, Hilden, German y), according to the manufactur er's pr otocol.The concentr ation of DN A w as quantified using Qubit ® 2.0 Fluorometer with DNA HS kits (Life Technologies , Carlsbad, C A, USA).16S rRNA gene amplicon sequencing was performed by Macrogen (Amsterdam, The Netherlands) using the Illumina MiSeq Next Generation Sequencing platform * .P air ed-end libr aries wer e constructed using the Illumina Herculase II Fusion DNA Pol ymer ase Nexter a XT Index Kit V2 (Illumina, Eindho ven, Netherlands).T he primers used for bacterial amplification were Bac341F (5 -CCTA CGGGNGGCWGCA G-3 ; (Herlemann et al. 2011 ) and Bac806R (5 -GGA CTA CHV GGGTWTCTAAT-3 ; (Caporaso et al. 2012 ).Ar chaeal amplification w as performed with primers Arch349F (5 -GYGC ASC AGKCGMGAAW-3 ) and Arch806R (5 -GGA CTA CVSGGGTATCTAAT-3 ; (Takai and Horikoshi 2000 ).Analysis of the 16S rRNA sequencing output files was performed within R version 3.5.1 (Team 2013 ) using the D AD A2 pipeline (Callahan et al. 2016 ).After c hec king the r eads quality of the samples, the length of the r eads wer e trimmed using the following parameter: truncLen = c(280 200).Taxonomic assignment of the reads was up to the species le v el when possible, using the Silva non-redundant database version 132 (Yilmaz et al. 2014 ).Count data were normalized to relative abundances.Further data analysis and the creation of amplicon sequence variant (ASV) tables at different taxonomical levels were performed using the phyloseq and microbiome pac ka ges (McMurdie and Holmes 2013 ).

Statistical approach
All statistical tests were executed in R, developed by the R Core Team ( 2013 ).We tested the data for normal distribution and assumptions for e v ery test executed.To test for differences in CH 4 emissions between treatments in the field, a linear model (LM) was used on the log-transformed data (CH 4 emissions being the dependent v ariable, and tr eatments + date the independent v ariables).Additionally, to test for differences between dissolved Fe(II) concentrations and changes in ratio 13/12 CO 2 between treatments, an analysis of variance (tw o-w ay ANOVA) combined with Tuk e y's post-hoc test was used.Furthermore, the difference in net CH 4pr oduction r ates and aer obic CH 4 -oxidation r ates between tr eatments was tested using a W elch' s T-test for unequal variance and a Student's T-test, r espectiv el y.To visualize and test linear relationships for the increase in 13/12 CO 2 we used a LM ( 13/12 CO 2 being the dependent variable, and days of incubation * treatment * treatment the independent variables).Potential CH 4 -oxidation and CH 4 -pr oduction r ates wer e calculated using the slope of the linear part of the potential CH 4 -oxidation curve divided by the absolute dry weight (g) of the incubated sediment, and corrected for time.Only the linear part of the slope was used to minimize the effects of substrate limitation and effects of (by)product inhibition on the calculation of potential CH 4 -oxidation rates.

Results and discussion
Fe(II)Cl 2 ad dition effecti v el y lo w er ed PO 4 3-concentr ations in the enclosures, lo w ering the PO 4 3-concentration from 2.2 ± 0.4 to 1 ± 0.4 μM in the surface water, and consider abl y boosted Fe con-centrations in sediment and water (Table 1 ).This is in line with pr e vious findings, wher e under favor able Fe: P r atios and r edox conditions Fe addition successfull y loc k ed P into lak e sediments (Wang and Jiang 2016 ).Here, in Fe(II)Cl 2 -tr eated enclosur es, up to 91% of the Fe was present in the form of Fe(III) (Kang et al. 2023 ).CH 4 emissions of the Fe(II)Cl 2 -treated enclosures were on average 3.5 times lo w er than those of unamended ( P < 0.001, r 2 = 0.43, df = 34, F = 6.9, LM; Fig. 2 ), wher e contr ol CH 4 emissions av er a ged 52 ± 78 mg CH 4 m −2 d −1 and Fe(II)Cl 2 -treated enclosures 15 ±14 CH 4 m −2 d −1 , suggesting that Fe(II)Cl 2 addition lo w ered CH 4 emissions.Phosphorus control has previously been found to lo w er CH 4 emissions through indirect effects of oligotrophication on the aquatic foodweb (Nijman et al. 2022 ).Additionally, we observed a general descending CH 4 emission intensity over time (Fig. 2 ), which is likely due to seasonality, where in colder months CH 4 emissions decr eases.Her e, we test if the effects on CH 4 emission can also r esult fr om dir ect effects on micr obial CH 4 -pr oduction and oxidation in the sediment.
The aerobic CH 4 -oxidation rates of Fe(II)Cl 2 -treated sediments (av er a ge: 2.4 ± 1.2 μmol CH 4 g −1 d −1 ) were not significantly different than rates observed in the control-enclosure sediments (aver a ge: 3.6 ± 0.7 μmol CH 4 g −1 d −1 ; P = 0.34, df = 5, t = 1.1;T-test) (Fig. 3 A), implying that aerobic CH 4 -oxidation played a minor role in lo w ering CH 4 emissions fr om the Fe(II)Cl 2 -tr eated enclosur es.Ho w e v er, sediments fr om the Fe(II)Cl 2 -tr eated enclosur es sho w ed ∼3 times lo w er net CH 4 -pr oduction r ate compar ed to the control sediments (F e(II)Cl 2 a verage: 0.53 ± 0.13 μmol CH 4 g −1 d −1 , contr ol av er a ge: 1.4 ± 0.52 μmol CH 4 g −1 d −1 , P = 0.05, df = 3.5 t = 2.8, T-test, Fig. 3 B and S1 A).Hence, given that: (i) sediment and water samples from Fe(II)Cl 2 -treated enclosures sho w ed enrichment in Fe(III) content, (ii) other potential electron acceptors wer e onl y found in low concentrations (Table 1 , and in Kang et al. 2023 ), and (iii) potential aerobic CH 4 -oxidation rates were com-  S2 ) (C) 16S rRNA gene sequencing data, "in situ" refers to the microbial community originating from the original enclosure sediment, whereas the other three samples are taken from the incubations at the end of the experiment from each serum bottle (day 130).
parable between control and F e(II)Cl 2 -treated enclosures , while in contrast net CH 4 -production rates-including potential anaerobic methane oxidation-wer e differ ent, we hypothesized that Fe-AOM played an important role in lowering the in situ CH 4 emissions.
We monitor ed AOM r ates and Fe(III) r eduction in incubations of enclosure sediments with and without Fe(III) addition.We found clear indications that the AOM-potential was higher in sediments originating from the Fe(II)Cl 2 -treated enclosures than in sediments originating from untreated controls, as seen by their significantl y higher 13/12 CO 2 r atio after incubation with 13 CH 4 (avera ge 13/12 CO 2 r atio at the end of the incubation period: control 0.22 ± 0.001, Fe(II)Cl 2 0.29 ± 0.002) (Fig. 4 A and S1 B, Table S1 ).Moreov er, the sim ultaneous addition of 13 CH 4 and Fe(III) to control sediments led to higher AOM-r ates compar ed to the 13 CH 4 -only addition, and also significantly boosted Fe(II)-production, suggesting that Fe-AOM is a k e y pathway (Fig. 4 B and S1C ).This finding is in line with a study on anaer obic methanotr oph bior eactors inoculated with pad d y soil, where ferrihydrite and 13 CH 4 addition also resulted in a rapid onset of 13 CO 2 production, indicating the occurrence of Fe-AOM (He et al. 2021 ).
Micr obial comm unity anal ysis r e v ealed the pr esence of Fecycling and CH 4 -cycling micr oor ganisms in the control and F e(II)Cl 2 -treated sediments , implying there is Fe-AOM-potential (Fig. 4 C).The presence of Fe(II)-oxidizing bacteria supports the hypothesis that Fe(II) originating from Fe(II)Cl 2 can be oxidized to F e(III), pro viding an electron acceptor for F e-AOM (Benz et al. 1998, Nielsen and Nielsen 1998, Oikonomidis et al. 2010 ).The archaeal community mainly consisted of methanogens, explaining the observed high CH 4 emissions.Known CHproducing taxa such as Methanosarcinales and Methanomicrobia also include members capable of CH 4 -oxidation via reverse methanogenesis , possibly in volved in Fe-AOM (Oni and Friedrich 2017 ).The increase in 13/12 CO 2 correlated to total Fe(II) concentrations ( P = < 0.0001).Fe(II)Cl 2 -treated sediments had a marginally higher 13/12 CO 2 compared to control sediment (LM, P = 0.05, r 2 = 0.76, df = 90, F = 30.1).
Methanoperedens species known to mediate both nitratedependent A OM and Fe-A OM (Legierse et al. 2023 ) were also present, albeit at low abundance (below 0.06% in both treatments).Additionally, in both control and Fe(II)Cl 2 -supplemented enclosure sediments, the methanogen Methanomassiliicoccaceae , was abundant (control 5%, Fe(II)Cl 2 7%), suggesting its involvement in Fe-AOM (He et al. 2021 ).Although the Fe(II)Cl 2 -treated sediments had a slightly higher abundance of Fe-cycling bacteria, the control sediments already contained Fe-oxidizers and Fereducers, indicating the functional capacity for Fe-AOM within the indigenous microbial community.This also explains the rapidcompared to Weber et al. ( 2017 )-onset of Fe-AOM in our incubations .Furthermore , the relative abundance of sulphide-oxidizing phototrophic bacteria such as Chlorobium was substantially lo w er in the Fe(II)Cl 2 -treated sediments (2.5%, compared to the control 5.7%) which was likely related to the lo w er availability of sulphides that may have precipitated in the form of iron sulphide minerals.
At the end of our 130-day incubation experiment the microbial community did not differ m uc h fr om the in-situ comm unity composition.This implies that the structure and composition of bacterial and arc haeal comm unities was persistent and repr esented envir onmental conditions.Additionall y, we found that the micr obial comm unity in both sediments had the potential to reduce Fe(III), as suggested by the 16S rRNA sequencing, and indicated by the increase in Fe(II) throughout the incubation experiment (Fig. 5 A) .The Fe(II)-pr oduction r ates wer e similar between H 2 O and 13 CH 4 tr eatment; whic h may be due to the activ e CH 4 suppl y caused by the sediments' high methanogenesis rates .F e(III)-reducers , mainly affiliating with Geobacteraceae were present at similar abundances in both types of sediment (2.2% in the control and 2.6% in the FeCl 2 -treated enclosure), ho w ever , irr espectiv e of the incubation treatment, in all the Fe(II)Cl 2 -treated sediments , the higher a vailability of F e(III) minerals , resulted in a significantly higher concentration of total F e(II) o ver time (LM, P = 0.003, r 2 = 0.80, df = 91, F = 37.7).Ho w e v er, part of the Fe(II) formation may also arise from anaerobic respiration of organic substr ates.Additionall y, we found that the headspace CH 4 concentration correlated with total F e(II) ( P = < 0.0001) concentrations , which is a proxy for Fe(III) r eduction.Mor eov er, irr espectiv e of the incubation treatment, total headspace CH 4 concentration was lower in Fe(II)Cl 2 -treated sediments (Fig. 5 B, LM, P = 0.008, r 2 = 0.72, df = 91, F = 25.8).This suggests that less CH 4 is released when there is more F e(III) reduction.T his hypothesis is furthermore strengthened by the significant correlation between 13/12 CO 2 and total Fe(II) concentration, caused by Fe(III) reduction (LM, P = < 0.0001).Fe(II)Cl 2treated sediments had a marginally higher 13/12 CO 2 compared to control sediments (Fig. 5 C, LM, P = 0.05, r 2 = 0.76, df = 90, F = 30.1),as a result of more 13 CH 4 oxidation .
In conclusion, we show that Fe(II)Cl 2 -treatment of eutrophic sediments in an urban pond led to significantly decreased aquatic CH 4 emissions, whic h was likel y facilitated by a r a pid onset of Fe-AOM, without substantiall y c hanging the nativ e micr obial community.This highlights that Fe-AOM can be important in freshwater sediments .T her efor e, in sediments wher e Fe(III) is the main alternativ e electr on acceptor, Fe(II)Cl 2 a pplication has the potential to combat eutrophication as well as CH 4 emissions, contributing to climate-smart water management.

Ac kno wledgements
We would like to thank Germa Verheggen, Roy Peters from the RU-Aquatic Ecology and Environmental Biology Department and Se-

Figur e 1 .
Figur e 1. Schematic o verview of the sampling design.Where 8 sediment cores originating from the enclosures were sliced for either oxic incubations or anoxic incubations, following different treatments.Source: Partly created using Canva by the authors.

Figure 2 .
Figure 2. Water-atmosphere CH 4 emissions from the enclosures in the field Y-axis is on log10 scale.Boxes show the median and first and third quartiles, whiskers indicate upper and lo w er quartiles, and dots indicate individual sampling points.CH 4 emissions from the Fe(II)Cl 2 -treated enclosures (N = 4) were significantly lo w er compared to controls (N = 4) based on a linear model (LM) ( P < 0.001, r 2 = 0.43, df = 34, F = 6.9).

Figure 3 .
Figure 3. Batch incubation of sediments from the Fe(II)Cl 2 -treated enclosures (blue) and the control enclosures (red).(A) Aerobic CH 4 -oxidation potential (calculated from the linear part of the trend) per g.dry weight sediment per day ( μmol CH 4 g −1 d −1 ).No significant difference was observed between treatments (T-test, P = 0.34, df = 5, t = 1.1).Boxes show the median and first and third quartiles, whiskers indicate upper and lower quartiles, dots indicate individual sampling points, and 'x' indicates the mean.(B) The net potential CH 4 -production rate of the sediment originating from differ ent enclosur es, expr essed as μmol CH 4 per g.dry weight sediment per day ( μmol CH 4 g −1 d −1 ).Sediments fr om contr ol enclosur es hav e a mar ginall y significant higher net CH 4 -production rate, compared to the sediments from the Fe(II)Cl 2 -treatment (T-test, P = 0.05, df = 3.5, t = 2.8).

Figure 4 .
Figure 4. Batch incubation of sediments from the Fe(II)Cl 2 -treated enclosures (blue) and the control enclosures (red).(A) Increase in 13/12 CO 2, with a significant difference between Fe(II)Cl 2 -treated sediments and control based on a LM ( P < 0.0001, r 2 = 0.88, df = 90, F = 69.5).Shaded areas show the 95% confidence interval.(B) Concentrations of dissolved Fe(II) (mM) at the end of the experiment (day 129).Boxes show the median and first and third quartiles, whiskers indicate upper and lo w er quartiles, dots indicate individual sampling points, and X indicates the mean.Letters depict which groups significantly differ from each other based on a tw o-w ay ANOVA ( P < 0.01, df = 2, F = 6.3) combined with a Tuk e y post-hoc test ( TableS2) (C) 16S rRNA gene sequencing data, "in situ" refers to the microbial community originating from the original enclosure sediment, whereas the other three samples are taken from the incubations at the end of the experiment from each serum bottle (day 130).

Table 1 .
Av er a ge nitr ate and tr ace metal concentr ations in surface w ater and the sediment top lay er (5 cm) for control and Fe(II)Cl 2tr eated enclosur es at the end of the enclosur e experiment (Nov 2021).